Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases RNAses, DNAses
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/882—Staphylococcus

Definitions

• the invention concerns the new type II restriction endonuclease SwaI, a process for its isolation and its use.
• Type II restriction endonucleases are endodeoxyribonucleases which are able to recognize and cleave particular DNA sequences. In this process one phosphodiester bridge in each polynucleotide strand of the target sequence is hydrolyzed. Type II restriction endonucleases are thus of value for the analysis of DNA molecules. Although type II restriction endonucleases are known which are specific for numerous DNA sequences, there is still a need for further type II restriction endonucleases which are specific for DNA sequences that up to now have not been recognized by any of the known restriction endonucleases. The object of the present invention is therefore to provide a new restriction endonuclease which is able to specifically recognize and cleave a sequence which has previously not been recognized by any such enzyme.
• the new restriction endonuclease according to the present invention which is denoted SwaI hereafter, has a temperature optimum at 25° C.
• the enzyme has good activity between pH 7.0 and pH 8.0 in 50 mmol/l Tris/HCl buffer with 1.0 mmol/l DTE (dithioerythritol), 10 mmol/l MgCl and 100 mmol/l NaCl.
• the pH optimum is at pH 7.5.
• the recognition sequence can be confirmed by the complete digestion of the DNA's of the viruses SV40 and adeno 2, of the phages lambda, T7 and phiX174 and of the phage derivative M13mp7 as well as of the plasmids pBR322 and pBR328. These DNA molecules are treated with SwaI.
• Table 1 shows a comparison of the cleavage specificity observed experimentally with a cleavage site specificity determined by a computer for an enzyme which recognizes the following sequence:
• the cleavage position within the recognition sequence of the enzyme can be determined on an M13 derivative having this recognition sequence at a distance of ca. 30-200 bases from the binding site of the universal sequencing primer (Messing, J. et al., (1981) Nucl. Acids Res. 9, 309-321).
• M13 derivative having this recognition sequence at a distance of ca. 30-200 bases from the binding site of the universal sequencing primer (Messing, J. et al., (1981) Nucl. Acids Res. 9, 309-321).
• At first sequence reactions according to the dideoxy chain-termination method (Sanger, F. et al., (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560-564, Messing, J. et al., (1981) Nucl. Acids Res. 9, 309-321) are carried out on the single-stranded DNA of the M13 derivative with the universal sequencing primer.
• the sequencing primer is radioactively labelled at the 5' end with T4-polynucleotide kinase and [ ⁇ - 32 P]ATP.
• a partially double-stranded DNA is prepared in a filling in reaction with DNA-polymerase I, (Klenow enzyme) and a deoxynucleotide triphosphate mixture of dATP, dCTP, dGTP and dTTP.
• This DNA whose newly synthesized strand is radioactively labelled at the 5' end, is cleaved with the restriction endonuclease SwaI.
• Half of the cleavage preparation is additionally treated with T4-DNA polymerase in the presence of a mixture of all four deoxynucleotide triphosphates in order to obtain blunt DNA ends.
• the analysis of the reaction products is carried out by electrophoresis on sequencing gels (8 mol/l urea, 5% polyacrylamide) and subsequent autoradiography. The results are interpreted according to Brown, N. L. and Smith, M. (Methods in Enzymology 65 (1980) 391-401).
• the position of the cleavage site is determined by a comparison of the distances of migration of the radioactively-labelled fragments with the sequencing ladder.
• the samples which were additionally treated with T4 DNA polymerase show a migration of the bands which is identical compared to the sample which was only cleaved with SwaI. This therefore shows that SwaI produces a blunt DNA end.
• the number of cleavage sites determined experimentally is identical to the number of cleavage sites for the sequence
• SwaI is preferably isolated by culturing microorganisms of the genus Staphylococcus, preferably of the species Staphylococcus warneri and isolating the enzyme from the cells.
• Staphylococcus warneri DSM 5872 is particularly preferred.
• microorganism Staphylococcus warneri is deposited at the German Collection for Microorganisms (DSM), Mascheroder Weg 16, 3300 Braunschweig, BRD and has the deposit number DSM 5872.
• Adeno 2 DNA is for example suitable as the substrate.
• the DNA fragments obtained are separated electrophoretically in agarose gels in buffer systems usually used for the fragment separation in the presence of ethidium bromide.
• the microorganisms used for the isolation of the enzyme grow aerobically in Brain Heart Infusion Medium from the Difco Company.
• the optimal conditions for growth are at a temperature of 37° C., and at a pH between 6.5 and 7.5.
• the doubling time is about 3 hours.
• the enzyme is isolated and purified by the usual chemical and mechanical methods such as by high pressure dispersion, ultrasound or enzymatic lysis.
• the cells are lysed by means of a French press.
• the further purification of the supernatant is preferably carried out by means of affinity chromatography and ion-exchange chromatography.
• Heparin-Sepharose CL-6B (Pharmacia) is for example suitable as the material for the affinity chromatography.
• Cellulose phosphate (Whatman) is for example suitable as the cation exchanger.
• Staphylococcus warneri DSM 5872 is cultured at 37° C. for 12-15 hours and is harvested at the end of the logarithmic phase. Brain Heart Medium (Difco) is used as the culture medium.
• the cell paste (30 g wet weight) is resuspended in 2.4 volumes buffer A (40 mmol/l Tris-HCl, pH 8.0, 0.1 mmol/l EDTA, 7 mmol/l 2-mercaptoethanol), which contains protease inhibitors. Subsequently the cells are lysed by passing them twice through a French press at 23000 lb/inch 2 and the precipitate is separated off. NH 4 Cl (final concentration 0.1 mol/l) is added to the supernatant. The nucleic acids are removed by Polymin precipitation. Subsequently the centrifuged supernatant is fractionated on a heparin-Sepharose column.
• buffer A 40 mmol/l Tris-HCl, pH 8.0, 0.1 mmol/l EDTA, 7 mmol/l 2-mercaptoethanol
• NH 4 Cl final concentration 0.1 mol/l
• a gradient of 0-1 mol/l NaCl is used for the elution. SwaI is found in the fractions between 0.4 and 0.6 mol/l NaCl.
• the active fractions are equilibrated against buffer B (40 mmol/l Tris-HCl, pH 8.0, 0.1 mmol/l EDTA, 7 mmol/l 2-mercaptoethanol, 10% (w/v) glycerol) and fractionated on a DEAE fast-flow column.
• a gradient of 0-0.5 mol/l NaCl is used for the elution.
• the active fractions are dialyzed against buffer B.
• the active fractions are pooled and dialyzed against storage buffer (20 mmol/l Tris-HCl, pH 8.0, 10 mmol/l 2-mercaptoethanol, 100 mmol/l NaCl, 0.1 mmol/l EDTA and 50% (v/v) glycerol).
• the solution is incubated for 1 hour at 25° C., cooled on ice and 5 ⁇ l of a terminating reagent consisting of 7 mmol/l urea, 20% (w/v) saccharose, 60 mmol/l EDTA and 0.01% (w/v) bromophenol blue is added. Subsequently a separation is carried out by electrophoresis in 1% agarose gels for 3-4 hours at 100 V. The bands obtained are identified by comparison with a DNA length standard.
• a terminating reagent consisting of 7 mmol/l urea, 20% (w/v) saccharose, 60 mmol/l EDTA and 0.01% (w/v) bromophenol blue

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• 1990
  • 1990-05-07 DE DE4014524A patent/DE4014524A1/de not_active Withdrawn
• 1991
  • 1991-04-30 DE DE59106111T patent/DE59106111D1/de not_active Expired - Fee Related
  • 1991-04-30 AT AT91106981T patent/ATE125866T1/de not_active IP Right Cessation
  • 1991-04-30 EP EP91106981A patent/EP0456086B1/de not_active Expired - Lifetime
  • 1991-05-06 US US07/695,936 patent/US5158878A/en not_active Expired - Lifetime
  • 1991-05-07 JP JP3101183A patent/JPH0667318B2/ja not_active Expired - Fee Related

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